1. Field of the Invention
This invention relates to immunoassays for the determination of iodothyronines in biological fluids such as serum or plasma. In particular, the present invention relates to competitive binding immunoassay methods, reagent means, and test kits for determining iodothyronines in unextracted samples of serum or plasma through the use of blocking or dissociating agents for the binding of iodothyronines by thyroxine binding proteins (TBP) present in such samples.
The iodothyronines have the general formula: ##STR1## wherein .beta..sup.1 and .beta..sup.2 are, independently, hydrogen or iodine. The principal iodothyronines of clinical interest are 3,5,3',5'-tetraiodothyronine (thyroxine; T-4) wherein .beta..sup.1 and .beta..sup.2 are both iodine; 3,5,3'-triiodothyronine (T-3, or simply "triiodothyronine") wherein .beta..sup.1 is iodine and .beta..sup.2 is hydrogen; 3,3',5'-triiodothyronine ("reverse T-3") wherein .beta..sup.1 is hydrogen and .beta..sup.2 is iodine; and 3,3'-diiodothyronine wherein .beta..sup.1 and .beta..sup.2 are both hydrogen. The quantitative determination of the concentration of the various iodothyronines, particularly the hormones T-4 and T-3, in the blood is of important significance in the diagnosis of thyroid disorders.
In the blood, nearly all of the circulating iodothyronines are complexed with various carrier proteins including albumin, thyroxine binding prealbumin and thyroxine binding globulin (TBG), such carrier proteins being generically referred to herein as thyroxine binding protein (TBP). In order to measure the concentration of the total amount of an iodothyronine in a blood sample, such as serum or plasma, the TBP-bound forms must be dissociated to an analytically significant degree and the resulting total free iodothyronine determined. The dissociation of iodothyronines from TBP, particularly TBG, was originally accomplished by an extraction process (U.S. Pat. No. 3,414,383). Under the current state-of-the-art, iodothyronines can be determined by immunoassay in unextracted samples through the use of compounds found empirically to block, and cause dissociation of, TBP binding. In current competitive binding iodothyronine immunoassays, a test sample is combined with reagents including an antibody to the iodothyronine to be determined, a labeled form (e.g., radiolabeled) of such iodothyronine, and one or more TBP blocking agents. The iodothyronine in the sample complexed with TBP is dissociated therefrom and competes with labeled iodothyronine for binding to the antibody. The proportion of labeled iodothyronine that becomes antibody-bound compared to that which remains unbound from antibody is dependent on the total concentration of the iodothyronine in the sample and is measurable in a wide variety of ways depending on the particular immunoassay technique employed.
2. Description of the Prior Art
Various compounds have been discovered as useful TBP blocking agents, including tetrachlorothyronine [Mitsuma et al, J. Clin. Endocrinol. Metab. 33:365 (1971)], diphenylhydantoin [Lieblich and Utiger, J. Clin. Invest. 50:60a (1971)], salicylate [Larson, Metab. 20:976 (1971)], and the various materials disclosed by Hollander (U.S. Pat. No. 3,928,553) and Chopra (U.S. Pat. No. 3,911,096), particularly 8-anilino-1-naphthalenesulfonic acid (ANS). The structures and general properties of the known TBP blocking agents vary over an extremely wide range. The properties critical to operability as a TBP blocking agent in immunoassays, i.e., the ability to sufficiently dissociate iodothyronines from TBP at concentration levels insufficient to cause significant inhibition of the antibody binding reaction, are generally considered unpredictable from purely structural comparisons, although some theories of TBG blocking have been propounded [Brown and Metheany, J. Pharm. Sci. 63:1214 (1974)].
Fenclofenac [2-(2,4-dichlorophenoxy)phenylacetic acid] is a diphenyl ether having antirheumatic activity that has been reported to interfere with thyroid function tests [Lancet 1:267 (Feb. 2, 1980), Lancet 1:432 (Feb. 23, 1980), Lancet 1:487 (Mar. 1, 1980), Capper et al, Clin. Chim. Acta 112:77(1981), and Kurtz et al, Clin. Endocrinol. 15:117(1981)]. Subsequent workers have raised the question whether fenclofenac would be suitable as a TBG blocking agent in thyroid function radioimmunoassays [Ratcliffe et al, Clin. Endocrinol. 13:569(1980)]. Capper et al, supra, also studied the effect of diclofenac [2-(2,6-dichlorophenylamino)phenylacetic acid].